Disposable chamber for analyzing biologic fluids

ABSTRACT

An apparatus for analyzing biologic fluid is provided that includes a first planar member, a second planar member, and at least three separators. At least one of planar members is transparent. The separators are disposed between the members, and separate the members to form a chamber having a height. At least one of the members or separators is sufficiently flexible to permit the chamber height to approximate the mean size of the separators. During use, the biologic fluid to be analyzed is disposed within the chamber.

This application is a divisional of U.S. patent application Ser. No.10/599,695 filed Oct. 5, 2006, which claims priority benefits under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/560,307filed Apr. 7, 2004, and PCT Patent Application No. PCT/US05/11602 filedApr. 7, 2005, the disclosures of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to chambers for analyzing biologic fluidsin general, and to chambers that permit the enumeration of particulatematter within the biologic fluid in particular.

2. Background Information

The classic method of enumerating particles in a liquid medium, such asblood cells in whole blood or bacteria or other material in urine orother biologic fluid is the hemocytometer, which includes a chambermanufactured to a precise height and having visible ruled areas ofprecise dimension. The liquid containing the particles to be enumeratedis introduced into the chamber. The liquid is diluted if necessary toreduce the number of particles to a manageable number. The operator thencounts the number of particles in a given demarcated area. Since thearea and height of the chamber are precisely known, the particle countper volume can be calculated. Although these chambers are generallyruled to demarcate a known area, this is not necessary if such a chamberis used in an image analyzer. With an image analyzer, rulings on thechamber itself are unnecessary because the field of view can be exactlycalculated from the image.

Because they are precisely manufactured, hemocytometer chambers arerelatively expensive and were not considered disposable. Modem precisionplastics molding techniques have allowed the manufacture of some typesof hemocytometer chambers at sufficiently low cost so as to beconsidered disposable in some instances, but chambers requiringsubstantial precision and/or thicknesses less than the traditional 0.1mm are very difficult to mold accurately.

U.S. Pat. No. 4,950,455 describes a counting chamber formed from a rigidglass slide and a rigid glass coverslip with rigid particles, such asglass beads, contained therebetween. The beads maintain a thin spacingbetween the slide and coverslip, thereby forming the counting chamber.

A counting chamber formed from rigid upper and lower panels separated byrigid particles has substantial limitations, however. Referring to FIGS.1 and 2, a prior art assembly generally denoted by 2 consists of a lowerglass slide 3, an upper glass coverslip 4 and an entrapped layer formedfrom a plurality of glass beads 5. Because any microscopic beads are notcompletely uniform, having a coefficient of variation of the diameter ofup to 10% or greater, the larger beads 6 “prop-up” the coverslip 4 tosome extent, and the smaller beads 7 have no effect on the separation.The differences in bead diameter is a problem because while it is easyto determine and/or control the mean diameter of the beads, the spreadof diameters is less well controlled, rendering the system less accuratethan is desired. This results in a separation between the upper andlower layers of about the mean bead diameter plus one standarddeviation. A greater problem is the presence of particulate debris asshown in FIG. 2. This debris can be present when the chamber is made orcan be introduced by the environment or from a sample. The debris 8 can“prop up” the coverslip 4 and create a large area of increased volume inthe chamber, which destroys its accuracy.

Another issue with this type of prior art chamber is that it isdifficult to package a plurality of such disposables in an instrumentused for automatically scanning and counting particles, such as an imageanalyzing system.

What is needed is an apparatus and method to overcome the limitations ofthe prior art, that provides a chamber for analyzing biologic fluids,including the enumeration of particulates within the fluid, which isinexpensive to produce, relatively insensitive to trapped particulatedebris, and amenable to packaging for use in an automated test system.

SUMMARY OF THE INVENTION

According to the present invention, an apparatus for analyzing biologicfluid is provided that includes a first planar member, a second planarmember, and at least three separators. At least one of planar members istransparent. The separators are disposed between the members, andseparate the members to form a chamber having a height. At least one ofthe members or separators is sufficiently flexible to permit the chamberheight to approximate the mean size of the separators. During use, thebiologic fluid to be analyzed is disposed within the chamber.

According to one aspect of the present invention, each planar member isa tape that can be wound on a reel. In some embodiments, the planarmembers are initially attached to one another. In other embodiments,each planar member is initially separated from the other planar member.

According to one aspect of the present invention, a cassette is providedhaving at least one source reel and at least one take-up reel. Theplanar members are initially wound on a source reel, and are transferredto a take-up reel during operation of the apparatus. An analysis regionis disposed between the source and take-up reels. The planar memberspass through the analysis region during the operation of the apparatus.

There are numerous advantages associated the present invention. Wediscovered that if a counting chamber is produced using separatorsdisposed between planar members, and if at least one the planar membersand separators is flexible, the chamber behaves differently than theprior art devices, and the difference is highly advantageous. When acounting chamber is filled with a liquid, the capillary forces tend topull the top and bottom planar members together, thus exerting a slightpressure on the retained separators. This pressure will cause theflexible element to deform in such a manner as to cause the chamberthickness to approximate, on average, the mean dimension of theseparators disposed between the planar members. For example, if both topand bottom planar members are rigid and the separators are flexible,separators larger than the mean diameter will be compressed, and theplanar members will approximate until more and more separators come intocontact with the planar members, preventing further approximation. Atthat point, the height of the chamber approximates the average height ofthe separators and is readily ascertainable. In another example, if theseparators are rigid and the top planar member is flexible, the topplanar member will deform and be “tented-up” in a small area around eachof the larger separators and be lower over smaller separators. Thechamber will have an average height which closely approximates averageseparator height.

An advantage of the present invention is, therefore, that a chamber isformed having a volume that is accurately determinable because theheight of the chamber is substantially uniform.

Another advantage of the present invention is that it can bemanufactured in an inexpensive form and still provide the desiredaccuracy. The present invention does not require accurately machinedvoids or separators to accurately establish volume. Consequently, theinvention can be manufactured inexpensively and still provide thedesired accuracy. In addition, because it can be manufacturedinexpensively, the present invention can practically be offered in adisposable form.

These and other objects, features and advantages of the presentinvention will become apparent in light of the detailed description ofthe invention provided below, and as illustrated in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The principles of the invention are further clarified by referring tothe following figures, where:

FIG. 1 is a cross-sectional schematic of the invention of the prior art,using a system in which all elements are rigid;

FIG. 2 is a cross-sectional schematic of the invention of the prior art,using a system in which all elements are rigid, and where particulatedebris has been trapped;

FIG. 3 is a cross-sectional schematic of the present invention, wherethe separators are flexible relative to the top and bottom planarmembers;

FIG. 4 is a cross-sectional schematic of the present invention, wherethe top planar member is flexible in relation to all other elements;

FIG. 5 is a cross-sectional schematic of the present invention, wherethe top planar member is flexible in relation to all other elements andwhere particulate debris has been trapped;

FIG. 6 is a schematic view of a first embodiment of the presentinvention;

FIG. 6A is a schematic view of an instrument designed to utilize asecond embodiment of the present invention;

FIG. 7 is a schematic view of a cassette containing the first embodimentof the present invention;

FIG. 8 is a schematic view of an instrument designed to utilize anembodiment of the present invention;

FIG. 9 is a schematic view of the instrument of FIG. 6 where the samplehas been added to the planar member;

FIG. 10 is a schematic view of the sample after spreading out betweenthe planar members; and

FIG. 11 is a schematic view of a typical field of view.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIGS. 3-11, the present invention apparatus 10 foranalyzing biologic fluid includes a first planar member 12, a secondplanar member 14, and at least three separators 16. At least one ofplanar members 12, 14 is transparent. The separators 16 are disposedbetween the members 12, 14, and separate the planar members 12, 14 toform a chamber 18 having a height 20. At least one of the members 12, 14or separators 16 is sufficiently flexible to permit the chamber height20 between the members 12, 14 to approximate the mean height of theseparators 16.

The separators 16 can be any structure that is disposable between theplanar members 12, 14, operable to space the planar members 12, 14 apartfrom one another. The dimension of a separator 16 that extends betweenthe planar members is referred to herein as the height 22 of theseparator 16. The heights 22 of the separators 16 typically do not equalone another exactly, but are within commercially acceptable tolerancefor spacing means used in similar analysis apparatus. Spherical beadsare an example of an acceptable separator 16 and are commerciallyavailable from, for example, Bangs Laboratories of Fishers, Ind., USA.

In some embodiments, the separators 16 consist of a material that hasgreater flexibility than one or both of the first planar member 12 andthe second planar member 14; i.e., relatively speaking, one or both ofthe planar members 12, 14 may be considered to be rigid relative to theseparators 16 and the separators 16 may be considered to be flexiblerelative to one or both of the planar members 12, 14.

In other embodiments, the separators 16 consist of a material that hasless flexibility than one or both of the first planar member 12 and thesecond planar member 14; i.e., relatively speaking, one or both of theplanar members 12, 14 may be considered to be flexible relative to theseparators 16 and the separators 16 may be considered to be rigidrelative to one or both of the planar members 12, 14.

Subject to the flexibility characteristics described above, the planarmembers 12, 14 can be made from a variety of materials, provided atleast one of the planar members 12, 14 is transparent. Transparentplastic films consisting of acrylic or polystyrene are examples ofacceptable planar members 12, 14. Planar members 12, 14 in the form of atape are particularly useful because they can be easily wound on a reel.

Now referring to FIG. 3, in an embodiment of the present invention 10the first planar member 12 and the second planar member 14 are separatedby a chamber 18 formed by plurality of separators 16 in the form ofspherical beads. These beads 16 are formed from a material that hasgreater flexibility than the first planar member 12 and the secondplanar member 14; i.e., the planar members 12, 14 may be considered tobe rigid relative to the beads 16 and the beads 16 may be considered tobe flexible relative to the planar members 12, 14. Plastic beads 16formed from polystyrene, polycarbonate, silicone and the like can beused. In this example, larger beads 16A are compressed to the pointwhere the planar members 12, 14 have approximated to the point wheremost beads 16 are touching the interior surfaces 24 of the planarmembers 12, 14, thereby making the chamber height 20 just slightly lessthan the mean bead diameter.

In FIG. 4, in another embodiment of the present invention 10 the firstplanar member 12 is formed from a material more flexible than thespherical beads 16 and the second planar member 14, and will overlay thebeads 16 in a tent-like fashion, where the areas between the beads 16are some arbitrary height determined by the bead diameters supportingthat piece of the first planar member 12. Any transparent plastic film,such as acrylic, polystyrene, or the like will work provided it is thinenough to flex as shown. It should be apparent that in thiscircumstance, although small local areas will deviate from the desiredchamber height 20, the average height of all the tented areas will bevery close to that of the mean bead diameter. Our testing indicates thatthat the mean chamber height can be controlled to 1% or better atchamber heights of less than four microns using the present invention.

FIG. 5 shows the chamber 18 of FIG. 4 wherein a piece of particulatedebris 26 has lodged. The first planar member 12 over the debris 26 hastented up, and the area under the debris 26 is of unknown height, butthis disturbance only affects a small area of the chamber 18, as opposedto what would occur if the whole system was rigid.

FIG. 6 shows another embodiment of the invention 10, where the secondplanar member 14 is formed from a one inch wide strip of transparentplastic film (e.g., polyethylene terphthalate (PET)) of approximatelyfifty (50) microns in thickness, the first planar member 12 is formedfrom the same material as the second planar member 14 but intwenty-three (23) micron thickness, and the chamber 18 therebetween isformed from a plurality of plastic beads 16 with a mean diameter of four(4) microns. The first planar member 12 has an inner coating of acoloration agent, such as acridine orange, which will differentiallycolor living white blood cells when examined with fluorescentillumination. Other reagents for fluorescence include astrozone orange,FITC, rhodamine and the like. Reagents which may be used withtransmitted light to differentially color the white blood cells includeastrozone orange, methylene blue, oxazine 170. The first planar member12 includes a plurality of ports 28 (e.g., approximately three hundred(300) microns in diameter) punched at regular intervals, and the planarmembers 12, 14 are bonded at some points 29 between the ports 28 to forma series of separated analysis chambers 18.

This spacing between the two planar members 12, 14 in this embodiment isaccomplished by spherical beads 16 of known and precisely controlleddiameter (e.g., about four (4) microns in diameter). These beads 16 arerandomly distributed on at least one of the planar members 12, 14 andcan be attached as part of the reagent film containing the stainingmaterial. The material retaining the beads 16 should be such that theyremain affixed to the planar member 12, 14 until at least after thefluid film movement has ceased so that they will not be swept away. Anacceptable method of coating a film with beads 16 is to suspend thebeads 16 in approximately a 0.5% solution of phytagel and apply a thincoating of the suspension by either spraying or meniscus coating. Theoptimum concentration of beads 16 will depend upon the type of bead andtheir method of manufacture, as well as the relative rigidity of the topand bottom planar members 12, 14. This concentration can be determinedempirically on a batch-to-batch basis by applying a series of beadconcentrations to the planar members 12, 14 to be used and then adding aliquid containing a dye, such as hemoglobin, which will give a usefuloptical density at the liquid layer thickness used. The average opticaldensity of the liquid layer is then plotted against bead 16 density todetermine the point where additional bead concentration produces nouseful change in liquid layer thickness; i.e., the point where thechamber height 20 is substantially uniform. An alternate means ofproviding the separators is to negatively emboss one of the planarmembers 12, 14 with projections having approximately the same height ofabout four (4) microns, for example by laser-etching pits in anip-roller and passing one planar member 12, 14 through the nip-rollerassembly.

FIG. 7 shows a cassette 30 having a shell 32 in which a source reel 34,a take-up reel 36, and a tape 38 extending therebetween are disposed.The “tape 38” is the embodiment of the present invention shown in FIG. 6and described above. Initially, the tape 38 is wound on the source reel34. Advancement of the tape 38 is controlled by rollers 40, which applytraction to the tape 38 at a point remote from the examination area 42and can act to draw the tape 38 from the source reel 34 as required. Thecassette 30 has a through-hole that allows an optical system to provideillumination through the tape 38.

FIG. 8 shows an optical analysis system 44 containing the cassette 32.The optical analysis system 44, which consists of joined componentsincluding a lens 46, a variable-wavelength light source 48 and a CCDcamera 50 are movable in three dimensions so as to allow the opticalsystem 44 to focus upon the tape 38 in the examination area 42 andprovide X-Y movement so as to allow scanning of the entire examinationarea 42, all under control of a system computer 52. Not shown is thesampling probe for extracting a biologic fluid (e.g., blood) from asample tube and depositing a small drop on the tape 38. This samplingdevice can take the form of a tube-piercing or similar probe, which usesa stepping motor-driven syringe to extract and deposit biologic fluidsamples. These devices are widely employed and well known to the art,and therefore will not be described further here.

FIG. 9 shows the assembly of FIG. 8 just after a drop of biologic fluid54 (e.g., blood) has been deposited into the sample entry port 28 (seeFIG. 6) of a chamber 18 foamed between the planar members 12, 14.

FIG. 10 is a schematic view of the entire area of the sample film 64 ofbiologic fluid 34, which generally has an irregular border. In thisexample, the biologic fluid is blood. Because the white blood cellswithin the sample film 64 tend to become readily entrapped in thechamber 18, they are generally found in highest concentration within afew millimeters of the port 28.

FIG. 11 is a schematic view of the analysis field 66 in FIG. 10, which,in the case of a whole blood sample, would show red blood cells 56,white blood cells 58, platelets 60, all surrounded by the blood plasma62. The beads 16 are also seen but are readily distinguished from allother elements because of their size and refractive index.

The characterization of the white blood cells 58 (white blood celldifferential count) is performed by the classification of eachindividual white blood cell 58 as it is encountered using eithertraditional image-processing methods or by the technique described inU.S. Pat. Nos. 5,321,975 and 6,350,613, both of which patents are herebyincorporated by reference. A number of supravital stains have beendescribed which differentially color the different classes of whiteblood cells 58 as has been described in U.S. Pat. No. 6,235,536, whichis also hereby incorporated by reference. Because the white blood cells58 are slightly compressed and readily imaged, stored images of cellsare viewable by the technologist in the case of questionable cellclassifications.

As an example of the utility of this invention, the white blood cell 58count of the sample film 64 may be performed by enumerating all of thewhite blood cells 58 found within the sample film 64 and dividing thatnumber by the volume of the sample film 64. Although it is possible todeposit a specific amount of sample within the chamber 18, it ispreferable to deposit an approximate amount and indirectly measure thevolume. This can be done by mechanisms such as: 1) the volume of thedrop of sample when first deposited can be calculated by interferometricimaging using optical techniques available from sources such as the ZygoCorporation of Middlefield, Conn. USA; or 2) the volume of samplefollowing film formation is calculated by measuring the area of the film64 and multiplying this by the average height of the film.

FIG. 6A shows an optical analysis system 44 containing anotherembodiment of the present invention 10 that includes a cassette 30 inwhich a second planar member reel 68, first planar member reel 70, andtake-up reel 72. Advancement of the planar members 12, 14 is controlledby take-up nip-rollers 74, which apply traction to the combined planarmembers 12, 14 at a point remote from the examination area 42 and canact to draw the planar members 12, 14 from their reels 68, 70 asrequired. The optical analysis system 44, which consists of joinedcomponents including a lens 46, a variable-wavelength light source 48and a CCD camera 50 are movable in three dimensions so as to allow theoptical analysis system 44 to focus upon the joined planar members 12,14 in the examination area 42 and provide X-Y movement so as to allowscanning of the entire examination area 42, all under control of asystem computer 52. A drop of biologic fluid 54 (e.g., blood) is showndeposited onto the second planar member 14. The nip-rollers 74 areoperable to advance the planar members 12, 14 to a point just past thenip-rollers 74, where the separators 16 disposed between the planarmembers 12, 14 are in contact with each planar member 12, 14, and thebiologic fluid contacts the interior surface 24 of each planar member12, 14 and spreads to form a thin sample film 64. The planar members 12,14 are then advanced so as to be readable by optical analysis system 44.

Since the overall accuracy of the system 44 when using a method ofvolume calculation depends upon the accuracy of the chamber height 20,it may be expedient to use an internal standard means to calculate theexact chamber height 20. An example of an internal standard includes aflexible or flowable material which is not miscible with the sample andwhich contains a known, stable and uniform concentration of a sensibleoptical dye. The material can be dyed flexible beads, dyed oil or thelike, and may be present in one or more areas of the chamber 18. Sincethe optical density is in direct proportion to the thickness of thecalibrator material, measurement of the optical density of the part ofthe calibrator material which completely fills the chamber height 20will allow the calculation of the exact chamber height 20 to within theprecision capabilities of the optical system.

Although the most frequent use for such a chamber 18 will be forenumerating blood cells in whole blood, it is equally useful forexamination of any undiluted fluid having sufficient particles to count.The chamber height 20 is not limited to the disclosed four microns butcan be larger or smaller to accommodate different separator sizes and/orconcentrations.

Although this invention has been shown and described with respect to thedetailed embodiments thereof, it will be understood by those skilled inthe art that various changes in form and detail thereof may be madewithout departing from the spirit and the scope of the invention.

1. A method of enumerating the cellular or particulate constituents of asample of whole, anticoagulated blood, comprising the steps of:providing an apparatus for analyzing biologic fluid that includes afirst planar member, a second planar member, wherein at least one of thefirst planar member and second planar member is transparent, and atleast three separators disposed between the planar members, eachseparator individually having a height and the separators collectivelyhaving a mean height, separating the planar members to form a chamberhaving a height extending between the planar members, wherein at leastone of the first planar member, second planar member, or separators issufficiently deformable to permit the chamber height to be substantiallyequal to the mean height of the separators; depositing a quantity ofbiologic fluid into contact with one of the first planar member orsecond planar member surface; approximating the planar members to form afilm of biologic fluid confined between the two planar members asseparated by the separators; determining the volume of biologic fluidcontained within the film; directly or indirectly enumerating allconstituents of interest within substantially the all of the film; andexpressing the enumerated constituents as a count per unit volume. 2.The method of claim 1, wherein the biologic fluid is blood.
 3. Themethod of claim 2, further comprising the step of: calculating thechamber height by measuring the average attenuation of light transmittedthrough the separators.
 4. The method of claim 3, wherein the step ofdetermining the volume of biologic fluid contained within the film,further comprises the steps of: determining the area of the film; andcalculating the volume of biologic fluid by multiplying the chamberheight times the area of the film.
 5. The method of claim 1 wherein thefilm volume is calculated by interferometric imaging of the drop ofbiologic fluid deposited onto the planar member prior to approximatingthe planar members.